Strong interaction of CpcL with photosystem I cores induced in heterocysts of Anabaena sp. PCC 7120

Phycobilisomes (PBSs) are photosynthetic light-harvesting antennae and appear to be loosely bound to photosystem I (PSI). We previously found unique protein bands in each PSI fraction in heterocysts of Anabaena sp. PCC 7120 by two-dimensional blue native/SDS-PAGE; however, the protein bands have not been identified. Here we analyzed the protein bands by mass spectrometry, which were identified as CpcL, one of the components in PBSs. As different composition and organization of Anabaena PSI-PBS supercomplexes were observed, the expression and binding properties of PBSs including CpcL to PSIs in this cyanobacterium may be diversified in response to its living environments.

Light-harvesting antennae have been diversified in photosynthetic organisms (Blankenship 2021) and can be grouped into membrane proteins and water-soluble proteins.Phycobilisomes (PBSs) are water-soluble pigment-protein complexes and consist of linker proteins and pigment-binding phycobiliproteins (MacColl 1998;Adir 2005;Watanabe and Ikeuchi 2013;Rast et al. 2019;Adir et al. 2020).PBSs are primarily bound to photosystem II of cyanobacteria and red algae as revealed by cryoelectron microscopy (Rast et al. 2019;You et al. 2023).However, it is unclear how PBSs interact with photosystem I (PSI) to catalyze light-induced electron transfer from plastocyanin or cytochrome c 6 to ferredoxin (Golbeck 1992;Brettel and Leibl 2001;Fromme et al. 2001;Nelson and Junge 2015).
Watanabe et al. purified PSI-tetramer-PBS supercomplexes from the cyanobacterium Anabaena sp.PCC 7120 (hereafter referred to as Anabaena) grown under normal conditions (Watanabe et al. 2014), whereas we have purified PSI-dimer-PBS and PSI-monomer-PBS supercomplexes from Anabaena grown under iron-deficient conditions (Shimizu et al. 2023).The two studies detected CpcL in the supercomplexes, which is one of the linker proteins in PBSs and was denoted by Watanabe et al. (2014).CpcL, previously known as CpcG3 in Anabaena, is one of the rod-core linker proteins (Bryant et al. 1991).It contains an N-terminal linker domain and a distinctive hydrophobic C-terminal domain (Kondo et al. 2007;Watanabe et al. 2014;Hirose et al. 2017;Guo et al. 2024).In contrast, CpcL was also called CpcG2 in the cyanobacterium Synechocystis sp.PCC 6803 and its characteristic protein structure has been determined by cryo-electron microscopy (Zheng et al. 2023).
It is known that CpcL of Anabaena was highly expressed in heterocysts (Watanabe et al. 2014), which are formed under nitrogen-starvation conditions (Wolk et al. 1994;Magnuson and Cardona 2016).We recently showed that by two-dimensional blue-native (BN)/SDS-PAGE, an apparent protein band in a molecular-weight range of 20.1-29.0 kDa was observed in the PSI fractions obtained from the heterocyst thylakoids of Anabaena, but this band was not observed in any PSI fractions from the vegetative thylakoids of Anabaena (Nagao et al. 2022).The protein band is presumed to be CpcL based on its apparent molecular weight; however, it has not yet been definitively identified.
The bands presumed to be CpcL in the two-dimensional BN/SDS-PAGE of the PSI tetramer and dimer fractions from the heterocyst thylakoids (labeled as red arrows in Figure 1A) were analyzed by mass spectrometry.The results showed that the N-and C-terminal polypeptides of CpcL can be detected clearly in both PSI tetramer and dimer fractions (Figure 1B-F).Because CpcL was hardly observed in any PSI fractions by two-dimensional BN/SDS-PAGE of vegetative thylakoids from Anabaena (Nagao et al. 2021;Nagao et al. 2022), strong interactions of CpcL with PSI cores appear to be induced in heterocysts.
This study demonstrated that CpcL is tightly bound to at least the PSI tetramer and dimer in the Anabaena heterocysts, and it was hardly observed in the PSI monomer fraction (Figure 1A).Under iron-deficient growth conditions of Anabaena, CpcL was detected from the PSI dimer and monomer fractions but not the tetramer (Shimizu et al. 2023).Thus, the association of CpcL with PSI oligomers may be changed under different conditions; e.g., iron-deficiency and nitrogen-starvation have induced a different association pattern of CpcL with PSI oligomers.It is interesting to note that any PBS components including CpcL were hardly detected in the PSI fractions from the vegetative thylakoids of Anabaena by two-dimensional BN/SDS-PAGE (Watanabe et al. 2011;Nagao et al. 2021;Nagao et al. 2022), although the PSI tetramer cores together with CpcL were fractionated from the vegetative thylakoids by a milder method of sucrose density gradient centrifugation (Watanabe et al. 2014).These observations suggest that the tight association of CpcL with PSI cores occurs in Anabaena grown under stress conditions, such as iron deficiency and nitrogen starvation.Thus, the expression and binding properties of PBSs including CpcL to PSIs in Anabaena may be diversified in response to its living environments.